Phosphoamino acid analysis to show phosphorylation of bcl-2 on serine. The figure shows 2D electrophoresis on thin layer chromatography with the label detected by autoradiography. The cells were incubated overnight with ³²P_i as controls or with ATRA or taxol. Cell lysates were prepared and immunoprecipitated with anti–bcl-2 antibody (6C8) and the immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membrane. After autoradiography, the radiolabeled bcl-2 band was excised, digested with TPCK-trypsin, lyophilized, treated with 6 N HCl for 1 hour at 110°C and dried. The phosphoamino acids were then identified by 2D electrophoresis first at pH 1.9 and then at pH 3.5. The positions of the radioactive amino acids were compared with ninhydrin-stained control amino acids, as indicated in each panel of the figure. The same methods were used to detect phosphorylation of amino acids obtained by in vitro labeling. Using both methods, only serine phosphorylation was detected.